No cures exist for patients with glioblastoma (GBM) due to the resistance of tumor cells to standard therapies. Stem-like tumor subpopulations seem especially refractory to most treatments, and it is becoming increasingly clear that specific tumor microenvironments can promote stem cell properties and chemoresistance. However, poor understanding of how emerging targeted therapies interact with other agents and the tumor microenvironment has limited their development. The long-term goal of the project is to develop Notch inhibitors as effective new therapies for glioblastoma and other malignant brain tumors. The objective of this proposal is to elucidate how Notch interacts with the tumor microenvironment and other treatments so pathway inhibitors can be effectively used in the clinic. The Notch pathway, which is required for generation and maintenance of non-neoplastic neural stem cells, also plays a key role in GBM cancer stem cells (CSC). It has been shown that the perivascular microenvironment promotes CSC through activation of Notch signaling, and a number of agent targeting blood vessels are currently in use. As tumor-associated blood vessels are removed, GBM and other tumors shift towards a hypoxic phenotype, and it is less clear how Notch will function in this microenvironment. The central hypothesis to be tested in this proposal is that Notch is a key mediator of GBM differentiation and therapeutic response not just in the perivascular niche, but also in the hypoxic microenvironment. Indeed, emerging data suggest that hypoxic tumor cells can recapitulate many of the molecular features which define the perivascular niche, and that Notch induces a stem-like phenotype and modulates the response to traditional chemotherapy in this context. The first two specific aims focus on understanding how Notch is activated in hypoxic GBM cells, and determining if Notch blockade can reverse the increase in CSC and treatment resistance promoted by hypoxia. The second two specific aims focus on the interaction between Notch inhibition, radiation, and temozolomide chemotherapy, and investigate a novel mechanism by which Notch blockade can sensitize GBM to this commonly used alkylating agent. These studies will determine how Notch activity is regulated in hypoxic glioma cells, and to establish a requirement for Notch in CSC induction and aggressive tumor behavior in the hypoxic microenvironment. They will also examine a novel epigenetic mechanism by which the pathway can modulate MGMT expression and temozolomide sensitivity. These results are all of high clinical relevance, and will have a direct impact on the development of a novel agent targeting CSC in glioblastoma.